Electron tomogrophy of mitochondria after the arrest of protein import associated with Tom19 depletion

In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induc ed point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA) . Using intermediate-voltage electron tomography, we have generated three-d imensional reconstructions of 28 FPA-treated mitochondria at four time poin ts (0-32 h) after the addition of FPA. We determined that the cristae surfa ce area and volume were lost in a roughly linear manner. A decrease in mito chondrial volume was not observed until after 16 h of FPA treatment. The in ner boundary membrane did not appear to shrink or contract away from the ou ter membrane. Interestingly, the close apposition of these membranes remain ed over the entire periphery, even after all of the cristae had disappeared . The different dynamics of the shrinkage of cristae membrane and inner bou ndary membrane has implications for compartmentalization of electron transp ort proteins. Two structurally distinct types of contact sites were observe d, consistent with recently published work. We determined that the cristae in the untreated (control) mitochondria are all lamellar. The cristae of FP A-treated mitochondria retain the lamellar morphology as they reduce in siz e and do not adopt tubular shapes. Importantly, the crista junctions exhibi t tubular as well as slot-like connections to the inner boundary membrane, persisting until the cristae disappear, indicating that their stability is not dependent on continuous protein import through the complex containing T om19.